All methods for phosphate rely on
the formation of a phospho -
molybdate complex and its subsequent
reduction to highly colored blue
compounds.
The procedure given below is taken
from Riley (Anal.
Chim. Acta, 27:31, 1962).
In this method the sample is allowed
to react with a composite reagent
containing molybdic acid, ascorbic
acid, and trivalent antimony.The resultant complex heteropoly acid is reduced in situ to give a blue solution the extinction of which is measured
at 885 nm.

Notes

Analysis should be performed as soon
as possible after sample collection
(0.5 - 2 hours).

Measurements should be taken within
2-3 hours after injection of
reagents.

Reagent blank should not exceed
0.02, if it does then the ammonium
molybdate is suspect.

If tubes are dried in an oven it
should not be done in same oven as
tubes used for Solarzano NH4
determination.

Dedicated volumetrics should be used
for each standard.

DI water direct from Barnstead
should be used for all reagents and
standards.

All glassware should be rinsed after
drying to remove any particulate
contamination.

General notes on using the
Spectrophotometer.Never remove cell during a run, it is not necessary to ever open the door
to the cell.
If there are bubbles in the line,
they can be removed by either
sipping air or cleaner.

1.Make sure Spec is set to sip 3ml.

2.
Set wavelength to 885 nm and make
sure the spec is reading absorbance.

3.
Make sure the waste tube enters the
phosphorus waste container.

4.
Sip DI water and 0 the reading.

5.
Read standards in order of
concentration (i.e. low standards
are read first (start w/ blanks and
work way up).
After each standard, sip water
through and re-zero if necessary.Sip water through between high standard and first sample to remove any
residual PO4.
Re-zero if necessary.

6.
Before re-zeroing, sip again to make
sure all residual material is
removed and the reading is stable.

7.
Wipe the sipper tube on spec w/
kimwipes to dry off the outside of
the tube in between samples so that
contamination is minimized.

8.
If absorbance is higher than highest
standards then you will need to
dilute your sample.

Standard Preparation

Stock A Solution-1000mM PO4-P

Dissolve 0.136 g
of anhydrous potassium dihydrogen
phosphate (KH2PO4,
m.w. = 136.07) in approximately
900ml of deionized water contained
in a 1 L volumetric flask (note all
salts should be dried in oven prior
to weighing).
Dilute the solution to the mark with
deionized water and mix it well.Transfer this solution to an amber
bottle.
Add 1ml chloroform for preservation.This solution is stable for many months.
Refrigerate when it is not in use.

Stock B Solution-100mM PO4-P

Prepare this
solution weekly.Using a volumetric pipet or a calibrated automatic pipet, add
10.0ml of Stock A to approximately
90 mL of deionized water contained
in a 100ml volumetric flask.
Dilute the solution to the mark with
deionized water and mix it well.Add 1ml chloroform for
preservation and store in an amber
bottle.

Working Standards

Prepare these
solutions daily.Use adjustable, micro liter pipettes to add the designated
volumes B listed in the following
table.Calibrate the pipet for each required volume.(NOTE:The
standards for each day are presented
in bold typeface.The other concentrations are included for reference, if needed.)Prepare each standard by adding the
required amount of stock to the
required volume volumetric flask
containing deionized water.
Standards for both Ammonium and
Phosphate can be made in the same
volumetrics.After adding standards for both methods, dilute each to the mark with
deionized water and mix well.
Keep these solutions tightly sealed.

Standard Concentration
(uM)

Total

Volume

(ml)

Calibrant

Volume

(ml)

Stock

Standard

0.05

100

0.05

B (100uM)

0.1

100

0.1

B

0.2

100

0.2

B

0.3

500

1.5

B

0.4

100

0.4

B

0.5

100

0.5

B

0.6

100

0.6

B

0.7

100

0.7

B

0.8

100

0.8

B

0.9

100

0.9

B

1.0

100

1.0

B

2.5

100

2.5

B

5.0

100

5.0

B

Record the absorbance for each standard and spike
sample in the nutrient log book and
in a spreadsheet to keep track of
trends and calculate standard
deviation mean detection limit.Determine the standard curve by plotting
absorbance (y-axis) versus standard
concentration (x-axis).We have created an Excel workbook, entitled nutrients.xls,
for data input.
Put a copy of the daily Excel
worksheet on a diskette and a copy
of the curves on the door to the
spec room.

The daily absorbance for each
should be fairly consistent from day
to day.If the absorbance is significantly different, there is a
problem with either the standards or
the stock solutions.Data from 1998 measurements show the following mean values
for each absorbance.

Standard

Mean

- 1 Std Dev

+ 1 Std Dev

Std Dev

Std error

Min

Max

Number

Blank

.001

-.001

.002

.001

.0002

-.001

.006

47

.05
mM

.006

.004

.008

.002

.0003

.003

.013

48

.1
mM

.012

.010

.014

.002

.0004

.009

.015

16

.3
mM

.029

.026

.032

.003

.0004

.025

.038

46

.4
mM

.040

.037

.043

.003

.0004

.036

.047

48

.7
mM

.071

.067

.075

.004

.001

.064

.079

48

1 mM

.102

.098

.106

.004

.001

.094

.112

48

Method Detection Limit

The detection limit for this method
should be determined daily.If the detection limits are consistent
for a couple of weeks, then it will
be necessary to perform this task
weekly.

Spike DI water with 2-3 times the
estimated instrument detection limit
.This should be around 0.2 to 0.3 mM.(Use the 0.2 or 0.3 mM standard as the spike.)
You must run at least 7 spikes as
samples after the standard curve has
been run.Fewer than 7 will result in the calculations being wrong.Calculate the method detection limit
(MDL) by

MDL= [t(7, 0.01)
* s]

where t=t statistic for 7 reps
(t=3.14) with 99% confidence and
s=standard deviation of the
calculated concentration.